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Extraction of Small Molecules from Fecal Samples and Testing of Their Activity on Microbial Physiology
从粪便样本中提取小分子并检测其对微生物的生理学活性   

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Applied and Environmental Microbiology
Jul 2017

Abstract

The human body is colonized by vast communities of microbes, collectively known as microbiota, or microbiome. Although microbes colonize every surface of our bodies that is exposed to the external environment, the biggest collection of microbes colonizing humans and other mammals can be found in the gastrointestinal tract. Given the fact that the human gut is colonized by several hundred microbial species, our group hypothesized that the chemical diversity of this environment should be significant, and that many of the molecules present in that environment would have important signaling roles. Therefore, we devised a protocol to extract these molecules from human feces and test their signaling properties. Potentially bioactive extracts can be tested through addition to culture medium and analyses of bacterial growth and gene expression, among other properties. The protocol described herein provides an easy and rapid method for the extraction and testing of metabolites from fecal samples using Salmonella enterica as a model organism. This protocol can also be adapted to the extraction of small molecules from other matrices, such as cultured mammalian cells, tissues, body fluids, and axenic microbial cultures, and the resulting extracts can be tested against various microbial species.

Keywords: Metabolome (代谢组学), Microbiome (微生物组学), Gut (肠道), Small molecules (小分子), Extraction (提取), Microbial signaling (微生物信号转导)

Background

Complex assemblages of microbes live in and on humans, colonizing every surface exposed to the external environment. These communities have received several denominations over the decades, including normal flora, microbiota, and, more recently, microbiome (Sekirov et al., 2010; Kashyap et al., 2017). In humans, these vast microbial communities colonize our skin, respiratory tract, genitals, gastrointestinal tract, and many other sites. By far, the most heavily colonized site is the gastrointestinal tract, where trillions of microbes, encompassing several hundred species, coexist peacefully with their hosts. Some of these species knowingly live in symbiotic associations with the human organism, where both parts benefit from the interactions. For others, the relationship may be purely commensal, where the parts coexist without causing any harm to each other, but without providing or obtaining any benefit (Sekirov et al., 2010; Kundu et al., 2017).

The human gastrointestinal microbiota presents significant diversity in their composition, and stands out as a complex environment where interactions between different microbes as well as between microbes and host cells constantly occur. Bacteria are known to produce a plethora of bioactive small molecules, such as antibiotics, bacteriocins, pigments, secondary metabolites, quorum sensing signals, and many others (Antunes and Ferreira, 2009; Antunes et al., 2010; Antunes et al., 2011a). In such a complex environment as the intestinal microbiome, it is almost imperative to consider the production and accumulation of such molecules. These small molecules may represent by-products of metabolic activities or signals with specific roles, and can be produced both by the host itself as well as the microbes living in that environment; in many cases these small molecules are the tools used by these organisms to interact. Using high-throughput mass spectrometry-based metabolomics, we have previously shown that thousands of small molecules can be found in the lumen of the mammalian intestinal tract, and that the gut microbiome is involved in the production of many of them (Antunes et al., 2011b). In order to ascertain the signaling potential of small molecules from the gut metabolome, we have also extracted these molecules and tested their ability to modulate growth and gene expression of an enteric pathogen. As shown by our previous results, Salmonella enterica serovar Typhimurium responds to these molecules, and modulates the expression of over one hundred genes in response to bioactive small molecules from human feces (Antunes et al., 2014). Interestingly, many of the genes regulated by the fecal extract are required for the pathogenesis of Salmonella, such as those involved in the invasion of non-phagocytic host cells. More recently, we were able to purify and identify small aromatic compounds as the culprits for the regulation of Salmonella genes by the human gut metabolome (Peixoto et al., 2017). Here, we describe in detail the methods used by our group to obtain small molecules from the human gut metabolome and test them against Salmonella for various biological activities. A workflow of the procedures described herein can be found in Figure 1.


Figure 1. Workflow of the procedures described in this protocol

Materials and Reagents

  1. Polypropylene container (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 193A )
  2. Aluminum foil
  3. Tape
  4. Graduated glass pipettes (Fisher Scientific, catalog number: 13-678-25E )
  5. 2-ml Safe-Lock tubes (Eppendorf, catalog number: 0030120094 )
  6. Syringes (Descarpack, catalog number: 0324501 )
  7. Conical tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 362694 )
  8. Axygen universal pipette tips (Corning, Axygen®, catalog number: T-200-C-L-R )
  9. Barrier tips (Fisher Scientific, catalog number: 02-707-430 )
  10. Syringe filter, 0.22-µm pore (KASVI, catalog number: K18-230 )
  11. Cuvettes (BRAND, catalog number: 759115 )
  12. Inoculation loop
  13. Borosilicate tubes, 16 x 100 mm (DWK Life Sciences, Kimble, catalog number: 73500-16100 )
  14. Bacterial culture (Salmonella enterica serovar Typhimurium SL1344)
  15. HPLC-grade ethyl acetate, ≥ 99.7% pure (Sigma-Aldrich, catalog number: 34858 )
  16. Nitrogen gas
  17. HPLC-grade methanol (Sigma-Aldrich, catalog number: 34860 )
  18. C18 cartridges containing 360 mg of sorbent (WATERS, catalog number: WAT051910 )
  19. Distilled water
  20. Phosphate-buffered saline (Sigma-Aldrich, catalog number: P5493-1L )
  21. RNeasy Mini Kit (QIAGEN, catalog number: 74106 )
  22. Agarose (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 17850 )
  23. QuantiTect Reverse Transcription Kit (QIAGEN, catalog number: 205311 )
  24. MinElute Reaction Cleanup Kit (QIAGEN, catalog number: 28204 )
  25. Primers (Integrated DNA Technologies)
  26. Power SYBR Green PCR Master Mix (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4367659 )
  27. Hydrochloric acid, 36.5-38% (Sigma-Aldrich, catalog number: H1758 )
  28. Sodium hydroxide, ≥ 98% pure (Sigma-Aldrich, catalog number: S8045 )
  29. Luria-Bertani (LB) Broth (BD, catalog number: 244620 )
  30. Bacteriological agar (Sigma-Aldrich, catalog number: A5306 )
  31. Streptomycin (Sigma-Aldrich, catalog number: S9137-25G )
  32. 1 N HCl (see Recipes)
  33. 1 N NaOH (see Recipes)
  34. LB broth or agar (see Recipes)
  35. LB with streptomycin (see Recipes)

Equipment

  1. Digital scale (Ohaus, model: SP202 )
  2. Glass bottle (Fisher Scientific, catalog number: FB800500 )
  3. Orbital shaker (BiomiXer, model: TS-2000A )
  4. Graduated glass cylinder (Laborglas, catalog number: 91376 )
  5. Glass boiling flask (Corning, PYREX®, catalog number: 4100-125 )
  6. Electronic pipette (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 9501 )
  7. Fume hood
  8. Rotary evaporator (Heidolph, catalog number: 560-01300-00 )
  9. 37 °C incubator
  10. Freezer, -20 °C
  11. P20 micropipette (Gilson, catalog number: F123600 )
  12. P200 micropipette (Gilson, catalog number: F123601 )
  13. P1000 micropipette (Gilson, catalog number: F123602 )
  14. Speed-Vac concentrator (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: SPD131DDA-115 )
  15. Vortex (Scientific Industries, model: Vortex-Genie 2, catalog number: SI-0236 )
  16. pH meter (Sigma-Aldrich, catalog number: MT 51302916 )
    Manufacturer: Mettler-Toledo, catalog number: 51302916 .
  17. Autoclave
  18. Visible range spectrophotometer
  19. Nucleic acid (UV) spectrophotometer
  20. Shaker (NOVATECNICA, model number: NT145 )
  21. Centrifuge for 96-well plates (Eppendorf, model: 5810 , catalog number: 5810000017)
  22. Real Time PCR Machine (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4376357 )

Software

  1. GraphPad Prism (GraphPad Software)
  2. StepOne Software v2.3 (Thermo Fisher Scientific)

Procedure

  1. Extraction of small molecules from fecal samples
    1. The donor should collect a fresh fecal sample using a clean–not necessarily sterile–polypropylene container, store it in the fridge, and bring it to the laboratory within the next 24 h.
    2. Once the fecal sample is in the laboratory, weight the sample using a digital scale.
      Note: Extracts can be prepared from samples of any weight. However, in order to achieve the desired concentration for testing, we recommend a minimum sample weight of 50 g.
    3. Transfer the sample to a clean glass bottle, not sterile.
    4. Based on the weight of the sample, add 1 volume of ethyl acetate into the glass bottle containing the sample.
    5. Close the bottle tightly, wrap it with aluminum foil, and place it horizontally in an orbital shaker.
    6. Fix the bottle to the shaker using tape.
    7. Incubate it overnight at room temperature, setting the orbital shaker to 50 rotations per minute.
    8. After the incubation, remove the bottle from the shaker, place it vertically on the bench and let it stand for 5 min, so that the solid material can precipitate.
    9. Carefully remove the solvent from the bottle using a glass pipette and transfer it to a new bottle.
    10. Measure and record the volume of solvent recovered using a graduated glass cylinder.
      Note: It is not possible to recover the entire volume of ethyl acetate used to perform this extraction. This is due to the fact that some of the solvent will be absorbed by the fecal sample.
    11. The extract can then be stored in a -20 °C freezer (Note 1).

  2. Drying the extract
    1. Transfer the desired volume of extract to a glass boiling flask or 2-ml Safe-Lock tubes using a glass pipette or Axygen pipette tips and a micropipettor.
    2. Completely evaporate the solvent using a rotary evaporator or a Speed-Vac concentrator (Note 2).
      Note: Because ethyl acetate has a low boiling point and some of the bioactive molecules may be temperature-sensitive, it is not recommended to increase the temperature on the evaporator or Speed-Vac above 37 °C.
    3. Store the dried extract in the original vial (a glass boiling flask or 2-ml plastic tubes) at -20 °C (Note 1).

  3. Purification of small molecules with potential bioactivity
    1. Thaw the dried extracts at room temperature.
      Note: Thawing the dried extract may produce some water precipitation. If this is the case, the precipitate can be dried by applying a gentle stream of nitrogen gas into the flask or tube.
    2. Add 1 volume of 25% methanol (in water) to the vial(s) containing the dried extract.
    3. Scrape the walls of the vial with a glass pipette or a pipette tip, pipetting up and down and thoroughly vortexing the vials periodically.
    4. Take the desired number of C18 cartridges and, using a syringe, apply 2 ml of methanol through each cartridge.
    5. Using a syringe, apply 2 ml of water through each cartridge.
    6. Apply a maximum of 5 ml of extract through each column.
    7. Collect the flow-through using a conical tube.
    8. Wash each column with 4 ml of distilled water.
    9. Elute the molecules bound to the resin using 2 ml of solutions of increasing methanol concentrations–concentrations should be determined by the user, we suggest using 30, 40, 50, 60, 70, 80, 90, and 100% methanol (in water).
    10. Collect each fraction using a conical or Safe-Lock tube.
    11. Completely evaporate the solvent using a Speed-Vac concentrator.
    12. Save fractions in a -20 °C freezer until testing (Note 1).

  4. Preparing culture medium containing the desired extract
    1. The desired extracts or fractions obtained above should be removed from the freezer and LB broth should be added to the tubes–the volume added will vary, but we recommend starting with the same volume of culture medium as the volume of solvent evaporated (1x concentration).
    2. Scrape the walls of the tubes with a glass pipette or a pipette tip, pipetting up and down and thoroughly vortexing the tubes periodically.
      Note: Some samples will not solubilize completely. Due to the nature of the solvents used, some extracts will have a high fat content. In some cases, a significant amount of the material will either stick to the tube wall or form insoluble flakes in the culture medium.
    3. Filter the culture medium using a syringe and a 0.22-µm-pore filter to remove debris and any insolubilized material.
    4. Measure the pH of the solution.
    5. Adjust the pH to approximately 7.3 using HCl or NaOH.
    6. Add streptomycin to achieve a final concentration of 100 µg/ml.
    7. Filter-sterilize the solution using a syringe and a 0.22-µm-pore filter.
    8. Save the medium at room temperature until used (for storage times longer than 48 h we recommend storing the medium in a 4 °C fridge) (Note 1).

  5. Testing the effect of small molecules from the fecal metabolome on bacterial growth
    1. Using an inoculation loop, take a small amount of bacterial culture from a frozen stock of Salmonella enterica serovar Typhimurium SL1344 and streak a plate of LB containing 100 µg/ml of streptomycin.
      Note: SL1344 can be obtained from the National Collection of Type Cultures, Public Health England, at https://www.phe-culturecollections.org.uk/collections/nctc.aspx.
    2. Incubate overnight at 37 °C.
    3. Using an inoculation loop, take a single colony of Salmonella and transfer it to approximately 2 ml of LB broth containing 100 µg/ml of streptomycin, in 16 x 100 mm borosilicate tubes.
    4. Incubate it overnight at 37 °C with shaking (225 RPM).
    5. Dilute the culture 1:10 using phosphate-buffered saline.
    6. Measure the optical density of the suspension and multiply the value obtained by 10 to achieve the optical density of the original suspension.
    7. Calculate the volume of culture required to inoculate 8 ml of culture medium and achieve a starting optical density at 600 nm (OD600) of 0.05.
    8. Inoculate the desired number of 16 x 100 mm borosilicate tubes containing 8 ml of either LB broth or LB broth to which the fecal extract had been added, and 100 µg/ml of streptomycin.
      Note: If previously stored at 4 °C, culture medium should be equilibrated to room temperature before inoculation with Salmonella.
    9. Incubate at 37 °C with shaking (225 RPM).
    10. Every 30 min, remove a small aliquot from each of the cultures and use it to measure and record the OD600 of the cultures.
      Note: After the initial time points, the cultures will need to be diluted in phosphate-buffered saline before measurements can be made. This should be done to make sure that the optical density values obtained fall within the linear range of the equipment. Please be mindful of the linear range of your specific spectrophotometer.
    11. Using GraphPad Prism, plot the values obtained in a curve where the y-axis represents the optical density values and the x-axis represents the time of incubation.
    12. Compare the curves to evaluate if the extract has any effect on bacterial growth–statistical analyses can be performed within GraphPad Prism.

  6. Testing the effect of small molecules present in the extract on bacterial gene expression
    1. Prepare a growth curve experiment exactly as described above, using either LB or LB containing a fecal extract–the number of replicates per condition can vary, but we recommend a minimum of 4 replicates per condition.
    2. Based on the growth curve previously performed using the same fecal extract, grow the cultures to the desired growth stage–we suggest that the cultures used for RNA extraction be in the exponential growth phase.
    3. Collect between 1-2 ml of each culture and transfer to a conical tube containing 2-4 ml (2 volumes) of RNAprotect Bacteria Reagent.
    4. Manually invert tubes 5 times to mix.
    5. Let it stand for 5 min at room temperature.
    6. Centrifuge at 5,000 x g for 5 min.
    7. Discard the supernatant.
      Note: At this stage, the pellet can be stored at -80 °C and the extraction can continue in the next few days.
    8. Extract RNA using the RNeasy Mini Kit, according to the manufacturer’s recommendations, using protocol 4 of the RNAprotect Bacteria Reagent Handbook (version 01/2015).
    9. Continue the extraction procedure using protocol 7 of the RNAprotect Bacteria Reagent Handbook (version 01/2015), including the on-column DNAse treatment.
    10. Determine the amount and concentration of the RNA obtained using a UV spectrophotometer.
    11. Test the samples for the presence of contaminant genomic DNA by polymerase chain reactions (PCR):
      1. Perform a regular PCR using Taq Polymerase and primers targeting the desired gene–we recommend using the housekeeping gene gapA (forward: GGCGCTAACTTTGACAAATACGAAGG, reverse: AGTCATCAGACCTTCGATGATGCCG; Peixoto et al., 2017).
      2. Use 1 ng of genomic DNA from Salmonella as a positive control and 100 ng of the RNA extracted for testing.
      3. Run the PCR products on a 1.5% agarose gel.
        Note: Stain the gel using your preferred method of nucleic acid staining.
      4. Observe the bands under UV light–the presence of a band of the expected size in the positive control and the absence of such band on the RNA samples tested indicates that no significant DNA contamination exists.
        Note: RNA should be stored at -80 °C while the presence of DNA in the samples is being tested.
    12. Synthesize complementary DNA (cDNA) using between 500-1,000 ng of RNA and the QuantiTect Reverse Transcription Kit, according to the manufacturer’s recommendations.
    13. Purify the cDNA using the MinElute Reaction Cleanup Kit, according to the manufacturer’s recommendations.
    14. Quantify the cDNA using a UV spectrophotometer.
    15. Dilute the cDNA so that all samples are at a concentration of 5-10 ng/µl.
    16. Perform Real-Time PCR using 1 ng of the cDNA as template, primers targeting the desired genes, and the Power SYBR Green PCR Master Mix.
    17. Determine the relative expression levels between samples grown in the absence or presence of the fecal extract using the ΔΔCt method, and the gapA gene as the housekeeping control.

Data analysis

  1. To analyze the effect of small-molecule extracts on bacterial growth, optical density values should be plotted in a curve where the y-axis represents the optical density values and the x-axis represents the time of incubation. This can be done using GraphPad Prism, and the average values should be plotted, together with the standard deviation or the standard error of means. The curves should be visually compared to evaluate if the small molecules have any effect on bacterial growth. For statistical purposes, we recommend a minimum of 4 replicates per condition and the use of two-tailed unpaired t-tests for the evaluation of statistical significance. In the unlikely event that values within one group of samples vary widely, outliers can be detected and removed using the Grubbs’ test (https://www.graphpad.com/quickcalcs/Grubbs1.cfm). Determining whether or not the extract has a remarkable effect on growth is somewhat subjective, but we suggest using a statistically-significant (P < 0.05), 25% (or higher) change on the optical density as a starting point.
  2. To analyze the effect of small-molecule extracts on bacterial gene expression, threshold cycle (Ct) values obtained from the Real-Time PCR equipment through the StepOne Software v2.3 should be processed using the ΔΔCt method. First, Ct values obtained with the housekeeping control gene (gapA is used here as an example) should be subtracted from the Ct values obtained with each the genes being tested. This should be done in a sample-specific manner, i.e., the gapA Ct from one sample should be subtracted from the test gene Ct of that same sample and no other. The resulting ΔCt should be transformed from its logarithmic form by applying the formula 2-ΔCt. The resulting values should then be normalized to the average of the values obtained in the control group. As a result, the average of the control group should be normalized to 1 and every single value from the control or test group should be compared accordingly. Results can be plotted as bars of the average values per group, with bars displaying the standard deviation or the standard error of means. As noted above, for statistical purposes, we recommend a minimum of 4 replicates per condition and the use of two-tailed unpaired t-tests for the evaluation of statistical significance. In case some values vary widely from the others within the same group of samples, outliers can be detected and removed using the Grubbs’ test (https://www.graphpad.com/quickcalcs/Grubbs1.cfm). As noted above, determining whether or not the extract has a significant effect can be subjective, and should be interpreted taking into account the experimental model used as well as the nature of the genes tested. Nevertheless, we suggest using a statistically-significant (P < 0.05), 2-fold change in gene expression as a starting point.

Notes

  1. The time the extract can be stored for will vary depending on the properties of each bioactive molecule. However, to avoid degradation we suggest that extracts be stored for a maximum of one month.
  2. The time required for complete evaporation will vary depending on the sample type, size, and water content. Solely as a reference, we expect that a sample divided into 1-ml aliquots will take about 1-2 h to completely evaporate in a Speed-Vac set at a temperature of 37 °C. A 50-ml sample in a rotatory evaporator set at the same temperature will take 2-4 h to fully evaporate.

Recipes

  1. 1 N HCl (100 ml)
    Add 3 ml of hydrochloric acid, 36.5-38% (Sigma-Aldrich) to 97 ml of distilled water
  2. 1 N NaOH (100 ml)
    Weigh 4 g of sodium hydroxide, ≥ 98% pure (Sigma-Aldrich) and add to 100 ml of distilled water
  3. LB broth or agar
    1. Weigh 25 g of LB powder and add 1 L of distilled water in a glass bottle
    2. For LB agar, add 15 g of agar to the solution
    3. Mix it well by shaking the bottle
    4. Measure the pH of the solution and adjust it to 7.3 using HCl or NaOH
    5. Sterilize the medium using an autoclave
  4. LB with streptomycin
    1. Weigh 100 mg of streptomycin
    2. Add 900 µl of water
    3. Mix thoroughly using a vortex
    4. Measure the volume obtained using a micropipettor and a pipette tip
    5. Add more distilled water to obtain a final volume of 1 ml
    6. Add 1 µl of this solution per 1 ml of LB broth or agar to obtain a final concentration of 100 µg/ml

Acknowledgments

This work was funded by the National Council for Scientific and Technological Development (CNPq-Brazil), the Rio de Janeiro State Funding Agency FAPERJ and the Ministry of Health of Brazil. The authors declare that no conflict of interest exists.

References

  1. Antunes, L. C. and Ferreira, R. B. (2009). Intercellular communication in bacteria. Crit Rev Microbiol 35(2): 69-80.
  2. Antunes, L. C., Ferreira, R. B., Buckner, M. M. and Finlay, B. B. (2010). Quorum sensing in bacterial virulence. Microbiology 156(Pt 8): 2271-2282.
  3. Antunes, L. C., Davies, J. E. and Finlay, B. B. (2011a). Chemical signaling in the gastrointestinal tract. F1000 Biol Rep 3: 4.
  4. Antunes, L. C., Han, J., Ferreira, R. B., Lolic, P., Borchers, C. H. and Finlay, B. B. (2011b). Effect of antibiotic treatment on the intestinal metabolome. Antimicrob Agents Chemother 55(4): 1494-1503.
  5. Antunes, L. C., McDonald, J. A., Schroeter, K., Carlucci, C., Ferreira, R. B., Wang, M., Yurist-Doutsch, S., Hira, G., Jacobson, K., Davies, J., Allen-Vercoe, E. and Finlay, B. B. (2014). Antivirulence activity of the human gut metabolome. MBio 5(4): e01183-01114.
  6. Kashyap, P. C., Chia, N., Nelson, H., Segal, E. and Elinav, E. (2017). Microbiome at the frontier of personalized medicine. Mayo Clin Proc 92(12):1855-1864.
  7. Kundu, P., Blacher, E., Elinav, E. and Pettersson, S. (2017). Our gut microbiome: the evolving inner self. Cell 171(7):1481-1493.
  8. Peixoto, R. J. M., Alves, E. S., Wang, M., Ferreira, R. B. R., Granato, A., Han, J., Gill, H., Jacobson, K., Lobo, L. A., Domingues, R., Borchers, C. H., Davies, J. E., Finlay, B. B. and Antunes, L. C. M. (2017). Repression of Salmonella host cell invasion by aromatic small molecules from the human fecal metabolome. Appl Environ Microbiol.
  9. Sekirov, I., Russell, S. L., Antunes, L. C. and Finlay, B. B. (2010). Gut microbiota in health and disease. Physiol Rev 90(3): 859-904.

简介

人体被巨大的微生物群体统称为微生物群体或微生物群体。尽管微生物在我们身体的每一个暴露于外部环境的表面上定殖,但人类和其他哺乳动物中最大量的微生物可以在胃肠道中找到。鉴于人类肠道已被数百种微生物物种繁殖,我们的团队假设这种环境的化学多样性应该是显着的,并且该环境中存在的许多分子将具有重要的信号传导作用。因此,我们制定了一个协议,从人类粪便中提取这些分子并测试其信号特性。可以通过添加培养基并分析细菌生长和基因表达以及其他性质来测试潜在的生物活性提取物。本文描述的方案提供了一种简便且快速的方法,用于使用肠道沙门氏菌作为模型生物体从粪便样品中提取和测试代谢物。该方案还可以适用于从其他基质如培养的哺乳动物细胞,组织,体液和无菌微生物培养物中提取小分子,并且可以针对各种微生物物种测试所得提取物。

【背景】复杂的微生物群落生活在人类和人类身上,将暴露于外部环境的每个表面都定殖。几十年来,这些社区已经获得了几个教派,其中包括正常的植物群,微生物群和最近的微生物群(Sekirov等人,2010年; Kashyap等人,2017年)。在人类中,这些巨大的微生物群落在我们的皮肤,呼吸道,生殖器,胃肠道和许多其他部位定殖。到目前为止,殖民地最多的地方是胃肠道,那里有数以万计的数万种微生物与其宿主和平共存。这些物种中的一些明知地与人体有共生关系,两部分都受益于相互作用。对于其他人而言,这种关系可能纯粹是共生的,部分并存而不会对彼此造成任何伤害,但没有提供或获得任何好处(Sekirov et al。,2010; Kundu 等人。,2017)。

人类胃肠道微生物群的组成显着多样化,并且作为复杂的环境脱颖而出,不同微生物之间以及微生物和宿主细胞之间的相互作用不断发生。已知细菌产生大量生物活性小分子,如抗生素,细菌素,色素,次级代谢物,群体感应信号等等(Antunes and Ferreira,2009; Antunes等人,2010, ; Antunes 等,2011a)。在肠道微生物组这样的复杂环境中,考虑这种分子的生产和积累几乎是必要的。这些小分子可以代表具有特定作用的代谢活动或信号的副产物,并且可以由宿主本身以及生活在该环境中的微生物产生;在许多情况下,这些小分子是这些生物体相互作用的工具。使用基于高通量质谱的代谢组学,我们先前已经表明,在哺乳动物肠道的腔中可以发现数千个小分子,并且肠道微生物组参与其中许多小分子的生成(Antunes et al。,2011b)。为了确定来自肠道代谢组的小分子的信号传导潜力,我们还提取了这些分子并测试了它们调节肠道病原体生长和基因表达的能力。如我们先前的结果所示,肠道沙门氏菌血清型鼠伤寒沙门氏菌响应于这些分子,并且响应于来自人类粪便的生物活性小分子而调节超过一百个基因的表达(Antunes等人, / em>,2014)。有趣的是,许多由粪便提取物调节的基因是沙门氏菌发病的必需条件,例如涉及侵入非吞噬宿主细胞的那些。最近,我们能够纯化和鉴定小的芳香族化合物,作为人类肠道代谢组调节沙门氏菌基因的罪魁祸首(Peixoto等人,2017)。在这里,我们详细描述了我们的小组用于从人类肠道代谢组获得小分子的方法,并针对各种生物活性测试它们对抗沙门氏菌。这里描述的过程的工作流程可以在图1中找到。

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图1.本协议中描述的程序的工作流程

关键字:代谢组学, 微生物组学, 肠道, 小分子, 提取, 微生物信号转导

材料和试剂

  1. 聚丙烯容器(Thermo Fisher Scientific,Thermo Scientific TM,目录号:193A)
  2. 铝箔
  3. 磁带
  4. 刻度玻璃移液器(Fisher Scientific,目录号:13-678-25E)
  5. 2毫升安全锁管(Eppendorf,目录号:0030120094)
  6. 注射器(Descarpack,目录号:0324501)
  7. 锥形管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:362694)
  8. Axygen通用移液器吸头(Corning,Axygen <\ sup>,目录号:T-200-C-L-R)
  9. 屏障提示(Fisher Scientific,目录号:02-707-430)
  10. 注射器过滤器,0.22微米孔径(KASVI,目录号:K18-230)
  11. 比色杯(品牌,目录号:759115)
  12. 接种循环
  13. 硼硅酸盐管,16×100毫米(DWK Life Sciences,Kimble,目录号:73500-16100)
  14. 细菌培养(鼠伤寒沙门氏菌肠炎沙门氏菌SL1344)
  15. HPLC级乙酸乙酯,纯度≥99.7%(Sigma-Aldrich,目录号:34858)
  16. 氮气
  17. HPLC级甲醇(Sigma-Aldrich,目录号:34860)
  18. 包含360mg吸附剂(WATERS,产品目录号:WAT051910)的C18子墨盒
  19. 蒸馏水
  20. 磷酸盐缓冲盐水(Sigma-Aldrich,目录号:P5493-1L)
  21. RNeasy迷你试剂盒(QIAGEN,目录号:74106)
  22. 琼脂糖(Thermo Fisher Scientific,Thermo Scientific TM,目录号:17850)
  23. QuantiTect反转录试剂盒(QIAGEN,目录号:205311)
  24. MinElute Reaction Cleanup Kit(QIAGEN,产品目录号:28204)
  25. 引物(整合DNA技术)
  26. Power SYBR Green PCR Master Mix(Thermo Fisher Scientific,Applied Biosystems TM,产品目录号:4367659)
  27. 盐酸,36.5-38%(Sigma-Aldrich,目录号:H1758)
  28. 纯度≥98%的氢氧化钠(Sigma-Aldrich,目录号:S8045)
  29. Luria-Bertani(LB)肉汤(BD,目录号:244620)
  30. 细菌琼脂(Sigma-Aldrich,目录号:A5306)
  31. 链霉素(Sigma-Aldrich,目录号:S9137-25G)
  32. 1 N HCl(见食谱)
  33. 1 N NaOH(见食谱)
  34. LB肉汤或琼脂(见食谱)
  35. LB与链霉素(见食谱)

设备

  1. 数字秤(奥豪斯,型号:SP202)
  2. 玻璃瓶(Fisher Scientific,目录号:FB800500)
  3. 轨道摇床(BiomiXer,型号:TS-2000A)
  4. 刻度玻璃圆筒(Laborglas,目录号:91376)
  5. 玻璃烧瓶(Corning,PYREX®,目录号:4100-125)
  6. 电子移液管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:9501)
  7. 通风橱
  8. 旋转蒸发器(Heidolph,目录号:560-01300-00)
  9. 37°C培养箱
  10. 冰柜,-20°C
  11. P20微量移液器(Gilson,目录号:F123600)
  12. P200微量吸管(Gilson,目录号:F123601)
  13. P1000微量吸管(Gilson,目录号:F123602)
  14. Speed-Vac浓缩器(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:SPD131DDA-115)
  15. 涡流(Scientific Industries,型号:Vortex-Genie 2,目录号:SI-0236)
  16. pH计(Sigma-Aldrich,目录号:MT51302916)
    制造商:梅特勒 - 托利多,产品目录号:51302916。
  17. 高压灭菌器
  18. 可见范围分光光度计
  19. 核酸(紫外)分光光度计
  20. 摇床(NOVATECNICA,型号:NT145)
  21. 用于96孔板的离心机(Eppendorf,型号:5810,目录号:5810000017)
  22. 实时PCR仪(Thermo Fisher Scientific,Applied Biosystems TM,目录号:4376357)

软件

  1. GraphPad Prism(GraphPad软件)
  2. StepOne软件v2.3(赛默飞世尔科技)

程序

  1. 从粪便样品中提取小分子
    1. 捐献者应该使用清洁的,不一定无菌的聚丙烯容器收集新鲜的粪便样本,将其存放在冰箱中,并在24小时内将其带到实验室。
    2. 一旦粪便样品在实验室中,使用数字秤称重样品。
      注意:可以从任何重量的样品中制备提取物。但是,为了达到测试所需的浓度,我们建议最小样品重量为50克。
    3. 将样品转移到干净的玻璃瓶中,而不是无菌。
    4. 根据样品的重量,将1体积的乙酸乙酯加入含有样品的玻璃瓶中。

    5. 紧紧关闭瓶子,用铝箔包裹,并将其水平放置在定轨摇床中。

    6. 使用胶带将瓶子固定在摇床上。

    7. 在室温下孵育过夜,将轨道振荡器设置为每分钟50转。
    8. 孵化后,将瓶子从振动筛中取出,垂直放在工作台上静置5分钟,以使固体物质沉淀。

    9. 使用玻璃吸管小心地将溶剂从瓶中取出并转移到新瓶中。
    10. 测量并记录使用刻度玻璃量筒回收的溶剂量。
      注意:不可能回收用于进行该提取的整个体积的乙酸乙酯。这是因为一些溶剂会被粪便样品吸收。
    11. 然后将提取物储存在-20°C的冷冻箱中(注1)。

  2. 干燥提取物
    1. 使用玻璃吸管或Axygen移液器吸头和微量移液器将所需体积的提取物转移至玻璃烧瓶或2-ml Safe-Lock管中。

    2. 使用旋转蒸发器或Speed-Vac浓缩器(注2)完全蒸发溶剂 注意:由于乙酸乙酯的沸点较低,某些生物活性分子可能对温度敏感,因此建议不要将蒸发器或Speed-Vac的温度提高到37°C以上。 br />
    3. 将干燥的提取物储存在-20°C的原瓶(玻璃烧瓶或2毫升塑料管)中(注1)。

  3. 纯化具有潜在生物活性的小分子

    1. 在室温下解冻干燥的提取物。
      注意:解冻干燥的提取物可能会产生一些水分沉淀。如果是这种情况,可以通过将温和的氮气流施加到烧瓶或管中来干燥沉淀物。

    2. 加入1体积的25%甲醇(水溶液)到装有干燥提取物的小瓶中。

    3. 用玻璃吸管或移液器吸头刮擦小瓶壁,上下吸移并周期性地彻底振荡小瓶。
    4. 取所需数量的C18子弹,并使用注射器在每个药筒中加入2ml甲醇。
    5. 使用注射器,通过每个墨盒应用2毫升的水。

    6. 每个色谱柱最多应用5毫升提取物。

    7. 使用锥形管收集流通物
    8. 用4 ml蒸馏水清洗每个色谱柱。
    9. 使用2ml增加甲醇浓度的溶液洗脱与树脂结合的分子 - 浓度应由使用者确定,我们建议使用30,40,50,60,70,80,90和100%甲醇(在水中) 。

    10. 使用锥形或安全锁管收集每个部分
    11. 使用Speed-Vac浓缩器完全蒸发溶剂。
    12. 将馏分保存在-20°C冷冻箱中直至测试(注1)。

  4. 准备含有所需提取物的培养基
    1. 应从冷冻机中取出上述所需的提取物或馏分,并将LB培养液加入管中 - 添加量会有所不同,但我们建议从与蒸发溶剂体积相同体积的培养基开始(1x浓度) 。

    2. 用玻璃吸管或移液管尖端刮擦管壁,上下吸移并周期性地彻底涡旋管。
      注意:有些样品不会完全溶解。由于所用溶剂的性质,一些提取物将具有高脂肪含量。在某些情况下,大量的物质会粘附在管壁上或在培养基中形成不溶性薄片。
    3. 使用注射器和0.22微米孔径的过滤器过滤培养基以去除碎屑和任何不溶物质。
    4. 测量溶液的pH值。
    5. 使用HCl或NaOH将pH调节至约7.3。
    6. 加入链霉素以达到100μg/ ml的最终浓度。

    7. 使用注射器和0.22微米孔过滤器过滤消毒溶液
    8. 将介质保存在室温下直至使用(存放时间超过48小时,我们建议将介质存放在4°C的冰箱中)(注1)。

  5. 检测粪便代谢组小分子对细菌生长的影响
    1. 使用接种环,从沙门氏菌沙门氏菌血清型鼠伤寒沙门氏菌SL1344的冷冻储液中取少量细菌培养物,并划出含有100μg/ ml链霉素的LB平板。
      注意:SL1344可以从英国公共卫生类型文化国家集合获得,其位于 https://www.phe-culturecollections.org.uk/collections/nctc.aspx

    2. 在37°C孵育过夜。
    3. 使用接种环,取一个沙门氏菌单菌落,并将其转移到大约2毫升含有100微克/毫升链霉素的LB肉汤中,在16×100毫米硼硅酸盐管中。

    4. 在37°C振荡培养过夜(225 RPM)。

    5. 用磷酸盐缓冲盐水稀释1:10的培养液
    6. 测量悬浮液的光密度并乘以10得到的值以获得原始悬浮液的光密度。
    7. 计算接种8ml培养基所需的培养物体积,并在0.05nm(OD 600)下达到0.05的起始光密度。
    8. 接种所需数量的16×100mm硼硅酸盐试管,其中含有8ml添加了粪便提取物的LB肉汤培养基或LB肉汤培养基和100μg/ ml链霉素。
      注意:如果事先储存在4°C,培养基应在接种沙门氏菌前平衡至室温。

    9. 在37°C振荡孵育(225 RPM)。
    10. 每30分钟,从每种培养物中取出一小份,并用它来测量和记录培养物的OD 600。
      注意:在初始时间点之后,培养物需要在磷酸盐缓冲盐水中稀释后才能进行测量。应该这样做以确保获得的光密度值落在设备的线性范围内。请注意您的特定分光光度计的线性范围。
    11. 使用GraphPad Prism绘制曲线中获得的值,其中y轴表示光密度值,x轴表示孵育时间。
    12. 比较曲线以评估提取物是否对细菌生长有任何影响 - 统计分析可以在GraphPad Prism中进行。

  6. 检测提取物中存在的小分子对细菌基因表达的影响
    1. 使用含有粪便提取物的LB或LB准备与上述完全相同的生长曲线实验 - 每种条件的重复次数可以变化,但我们建议每个条件至少重复4次。
    2. 基于之前使用相同粪便提取物进行的生长曲线,将培养物培养至所需的生长阶段 - 我们建议用于RNA提取的培养物处于指数生长阶段。
    3. 收集1-2ml的每种培养物并转移到含有2-4ml(2体积)RNAprotect Bacteria Reagent的锥形管中。

    4. 手动翻转管5次混合。
    5. 让它在室温下静置5分钟。

    6. 在5,000 em x g离心5分钟。
    7. 丢弃上清液。
      注意:在此阶段,沉淀可以储存在-80°C,并且在接下来的几天内可以继续提取。
    8. 根据制造商的推荐,使用RNeasy Mini Kit提取RNA,使用 RNAprotect Bacteria Reagent Handbook(version 01/2015)
    9. 按照 RNAprotect Bacteria Reagent Handbook (01/2015版),包括柱上DNA酶处理。
    10. 确定使用紫外分光光度计获得的RNA的量和浓度。
    11. 通过聚合酶链式反应(PCR)测试样品中是否存在污染基因组DNA:
      1. 使用Taq聚合酶和靶向所需基因的引物进行常规PCR - 我们推荐使用看家基因gapA(正向:GGCGCTAACTTTGACAAATACGAAGG,反向:AGTCATCAGACCTTCGATGATGCCG; Peixoto等人,2017年) )。
      2. 使用1 ng来自沙门氏菌的基因组DNA作为阳性对照,并提取100 ng用于检测的RNA。
      3. 在1.5%琼脂糖凝胶上运行PCR产物。
        注意:使用您首选的核酸染色方法对凝胶进行染色。
      4. 在紫外灯下观察条带 - 在阳性对照中存在预期大小的条带,并且在测试的RNA样品上不存在这样的条带表明没有显着的DNA污染存在。
        注意:在测试样品中存在DNA时,RNA应储存在-80°C。
    12. 根据制造商的建议,使用500-1,000 ng的RNA和QuantiTect Reverse Transcription Kit合成互补DNA(cDNA)。
    13. 根据制造商的建议,使用MinElute Reaction Cleanup Kit纯化cDNA。
    14. 使用紫外分光光度计定量cDNA。
    15. 稀释cDNA,使所有样品的浓度为5-10 ng /μl。
    16. 使用1 ng cDNA作为模板,靶向所需基因的引物和Power SYBR Green PCR Master Mix进行Real-Time PCR。
    17. 使用ΔΔCt方法确定在不存在或存在粪便提取物的情况下生长的样品之间的相对表达水平,并使用gapA基因作为家政对照。

数据分析

  1. 为了分析小分子提取物对细菌生长的影响,应将光密度值绘制在y轴代表光密度值并且x轴代表孵育时间的曲线中。这可以使用GraphPad Prism完成,平均值应与标准偏差或平均值的标准误差一起绘制。应该直观比较曲线,以评估小分子是否对细菌生长有任何影响。出于统计目的,我们建议每个条件至少重复4次,并使用双尾不成对的 t - 测试来评估统计显着性。如果一组样本中的数值差别很大,那么可以使用Grubbs的测试检测并移除异常值。(https://www.graphpad.com/quickcalcs/Grubbs1.cfm )。确定提取物是否对生长具有显着影响是有些主观的,但我们建议使用统计学显着的(<0.05),光密度的25%(或更高)的变化作为一个起点。
  2. 为了分析小分子提取物对细菌基因表达的影响,应使用ΔΔCt方法处理通过StepOne软件v2.3从实时PCR设备获得的阈值循环(Ct)值。首先,应从每个被测基因获得的Ct值中减去用管家控制基因获得的Ct值(这里使用 gapA 作为例子)。这应该以样本特定的方式完成,即 , ,应从相同样本的测试基因Ct中减去来自一个样本的gapA Ct,而不是其他样本。得到的ΔCt应该通过应用公式2 <-ΔCt从其对数形式转换而来。然后将得到的值标准化为对照组的平均值。因此,对照组的平均值应该归一化为1,并且应该相应地比较来自对照组或试验组的每一个值。结果可以绘制成每组平均值的条形图,条形图显示标准偏差或均值的标准误差。如上所述,为统计目的,我们建议每个条件至少重复4次,并使用双尾不成对的 t - 测试来评估统计显着性。如果某些值与同一组样本中的其他值存在很大差异,则可以使用Grubbs的测试( https://www.graphpad.com/quickcalcs/Grubbs1.cfm )。如上所述,确定提取物是否具有显着效应可能是主观的,并且应该考虑到所使用的实验模型以及所测试的基因的性质来解释。尽管如此,我们建议使用基因表达的统计显着性( <0.05),2倍变化作为起点。

笔记

  1. 提取物可以储存的时间取决于每种生物活性分子的性质。但是,为避免降解,我们建议将提取物储存最多一个月。
  2. 完全蒸发所需的时间取决于样品类型,尺寸和含水量。仅作为参考,我们预期分成1毫升等分试样的样品需要约1-2小时才能完全蒸发,温度设定在37°C。在旋转蒸发器中设置相同温度的50毫升样品需要2-4小时才能完全蒸发。

食谱

  1. 1 N HCl(100毫升)

    加入3 ml盐酸36.5-38%(Sigma-Aldrich)至97 ml蒸馏水
  2. 1 N NaOH(100毫升)
    称取4 g纯度≥98%的氢氧化钠(Sigma-Aldrich)并加入100 ml蒸馏水中
  3. LB肉汤或琼脂
    1. 称取25克LB粉末,并在玻璃瓶中加入1升蒸馏水。
    2. 对于LB琼脂,将15克琼脂加入到溶液中。
    3. 通过晃动瓶子来充分混合
    4. 测量溶液的pH值并使用HCl或NaOH将其调整至7.3。
    5. 使用高压灭菌器灭菌培养基
  4. LB与链霉素
    1. 称重100毫克链霉素
    2. 加900μl水
    3. 使用涡流彻底混合
    4. 测量使用微量移液器和移液枪头获得的体积
    5. 添加更多的蒸馏水,以获得1毫升的最终体积
    6. 每1ml LB肉汤或琼脂加入1μl该溶液以获得100μg/ ml的终浓度

致谢

这项工作得到了国家科学和技术发展委员会(CNPq-巴西),里约热内卢州政府资助机构FAPERJ和巴西卫生部的资助。作者声明不存在利益冲突。

参考

  1. Antunes,L.C.和Ferreira,R.B.(2009)。 细菌间的细胞间通讯 Crit Rev Microbiol 35( 2):69-80。
  2. Antunes,L. C.,Ferreira,R. B.,Buckner,M. M.和Finlay,B. B.(2010)。 细菌毒力的群体感应 微生物学 156(Pt 8):2271-2282。
  3. Antunes,L. C.,Davies,J. E.和Finlay,B. B.(2011a)。 胃肠道中的化学信号传导 F1000 Biol Rep 3:4.
  4. Antunes,L.C.,Han,J.,Ferreira,R.B.,Lolic,P.,Borchers,C.H。和Finlay,B.B.(2011b)。 抗生素治疗对肠道代谢组的影响 抗菌剂Chemother em> 55(4):1494-1503。
  5. Antunes,LC,McDonald,JA,Schroeter,K.,Carlucci,C.,Ferreira,RB,Wang,M.,Yurist-Doutsch,S.,Hira,G.,Jacobson,K.,Davies,J.,Allen -Vercoe,E.和Finlay,BB(2014)。 人类肠道代谢组的抗微生物活性 MBio 5 (4):e01183-01114。
  6. Kashyap,P.C.,Chia,N.,Nelson,H.,Segal,E。和Elinav,E。(2017)。 个性化医学前沿的微生物群。 Mayo Clin Proc 92(12):1855-1864。
  7. Kundu,P.,Blacher,E.,Elinav,E.和Pettersson,S.(2017)。 我们的肠道微生物群:不断发展的内在自我 Cell 171(7):1481-1493。
  8. Peixoto,RJM,Alves,ES,Wang,M.,Ferreira,RBR,Granato,A.,Han,J.,Gill,H.,Jacobson,K.,Lobo,LA,Domingues,R.,Borchers,CH, Davies,JE,Finlay,BB和Antunes,LCM(2017)。 抑制沙门氏菌宿主细胞侵入人类粪便中的芳香族小分子代谢组。 Appl Environ Microbiol 。
  9. Sekirov,I.,Russell,S.L.,Antunes,L.C和Finlay,B.B.(2010)。 健康与疾病中的肠道菌群 Physiol Rev 90 (3):859-904。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Alves, E. S., Ferreira, R. B. and Antunes, L. M. (2018). Extraction of Small Molecules from Fecal Samples and Testing of Their Activity on Microbial Physiology. Bio-protocol 8(8): e2808. DOI: 10.21769/BioProtoc.2808.
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